During the past century, many species of the Spotted Fever Group Rickettsia (SFGR)have been described, especially, through the introduction of a variety of molecular techniques applied to detect rickettsiae inside of their host. In this study we developed a quantitative realtime polymerase chain reaction (qPCR) assay (1) to characterize the growth and the distribution of a SFGR of unrecognized pathogenicity in naturally infected Amblyomma americanum ticks during physiological events; and (2) to terminate the influence of the host cell specificity in the replication patterns of recognized and unrecognized SFGR during a reciprocal rickettsiae challenge in both mammalian and tick cell lines. Rickettsia amblyommii was identified in the tissue samples of naturally infected A. americanum ticks at ratios of ¡Ü 1 rickettsiae per tick cell. Significant variability in the ratio of ickettsial to tick gene copy numbers between the tissues was identified; however, no single tissue was consistently observed to have the greatest rickettsial burden throughout the feeding event. Furthermore, the ratio of rickettsial to tick gene copy numbers did not significantly differ between eggs, immature ticks, and feeding events. In the in vitro study, differences in the ratio of rickettsiae per cell were observed within each cell line. The ratio of rickettsiae per host cell was greatest in Rickettsia-infected ISE6 cells, compared to Vero cells. Rickettsia parkeri infection load was consistently greater in both cell lines compared to R. amblyommii and Rickettsia montanensis; and considerable variability between these last two Rickettsia species was observed when the ratio of ickettisae per host cell was calculated for each individual cell line. The implications of the use of this technique to understand the pathogenic nature of some SFGR and to investigate the host specificity in the tick-SFGR interactions is further presented and discussed.

Several members of the spotted fever group (SFG) of Rickettsia are transmitted to the host through infected ixodid (hard) ticks, which can serve as both vectors and reservoirs. Multiple studies have demonstrated that ticks secrete proteins into the bite site of the host that suppress innate and adaptive immune responses. While this suppression of immune responses is beneficial to the tick, it may also be beneficial to the transmitted Rickettsia. We hypothesize that Rickettsia utilize the ticks ability to alter the host immune response at the tick feeding site to successfully establish infection. In the current study, we analyzed how the tick transmission influenced the response to Rickettsia infection by comparing the innate immune response following intradermal versus tick-inoculation of R. amblyommii in the skin. In particular, we analyzed mRNA expression of Toll-like receptor (TLR) and TLR adaptor/effector genes as well as proinflammatory cytokines and chemokines. Data was analyzed using One-way ANOVA and Newman-Keuls post tests. The results of this study are the first to delineate the immunomodulatory milieu associated with rickettsial infection during the natural route of tick exposure.

Rickettsia felis is a gram-negative bacterium predominantly described in the cat flea, Ctenocephalides felis. Since first described in 1990 in a commercial cat flea colony in the United States, R. felis has been detected in numerous arthropod species in 28 countries around the world. Additionally, as the etiologic agent of flea-borne rickettsiosis, R. felis can cause disease in humans, with patients presenting with clinical symptoms typical of rickettsial diseases including: fever, headache, and myalgia. Transmission of R. felis within flea colonies is predominantly via vertical (transovarial and transstadial) transmission and mechanisms of horizontal transmission are undescribed. Studies are needed to describe both arthropod and vertebrate determinants of R. felis horizontal transmission. Here we describe the development of both arthropod and vertebrate models of R. felis infection and use the tools of molecular biology to characterize R. felis infection in both models. We first characterized R. felis-infection in a naturally R. felis-infected cat flea colony and observed that the prevalence of R. felis-infection within a cat flea colony is dynamic with an inverse relationship between R. felis-infection density and prevalence of R. felis-infection in the colony. Also, over the flea lifespan, R. felis infection remains steady with little fluctuation during the onset of flea bloodmeal acquisition and oogenesis. After characterizing R. felis replication in naturally infected fleas, we developed a biological assay to infect naïve fleas. This is the first demonstration of oral acquisition and persistent R. felis-infection of fleas fed an R. felis-infected bloodmeal. Lastly, we describe the initial results of a murine model of R. felis infection. In this model, R. felis efficiently disseminated in the mouse and is detectable in several tissues including the spleen and liver for up to 14-days post-inoculation. Elucidation of both arthropod and vertebrate determinant for R. felis transmission is necessary to understand the ecology of R. felis in nature.

Edwardsiella ictaluri encodes a type III secretion system (T3SS) required for intracellular replication. Analysis of in vitro gene expression indicates the T3SS translocon proteins express and secrete in acidic pH. Expression of other T3SS genes, however, requires that phosphate be limited in the culture media, in addition to acidic pH. Responses to environmental stimuli are mediated through the T3SS-encoded regulatory proteins EsrA, EsrB, and EsrC. Mutations in these genes result in differing phenotypes. Mutation of EsrA results in moderately reduced expression of T3SS genes, but translocon protein secretion is retained in the mutant. However, the EsrA mutant is attenuated intracellularly and in vivo. Mutation of EsrB results in severely decreased T3SS gene expression and translocon protein secretion, leading to intracellular and in vivo attenuation. EsrB is also required for expression of type VI secretion system (T6SS)-related proteins through the modulation of EsrC expression. EsrC mutation has an effect on T3SS gene expression, but less so than EsrB, and does not abolish T3SS translocon secretion. EsrC mutation, however, does inhibit expression of T6SS-related proteins, indicating one of its functions is to coordinate expression of the T6SS with that of the T3SS. The EsrC mutant is not attenuated intracellularly, but does exhibit attenuated virulence in vivo. Expression of the T3SS also is dependent on two plasmids carried by strains of E. ictaluri virulent for channel catfish. Mutation of both of the plasmids results in severe decreases in T3SS gene expression, resulting in attenuation of E. ictaluri intracellularly and in vivo. The plasmids do not encode proteins with homology to known regulatory proteins, but may integrate into the genome near putative regulatory genes, thereby modulating their expression. The results presented here demonstrate that E. ictaluri responds to conditions mimicking the intracellular environment by expression a T3SS, which is required for intracellular survival. The expression of this T3SS absolutely is dependent on EsrB, and EsrA and EsrC are required for optimal T3SS expression.

First described in 1967, Isospora amphiboluri has become recognized as an important pathogen of bearded dragons. To date, no cross-sectional, pathogenesis, or treatment study has been performed to shed light on the epidemiology or treatment of I.amphiboluri. Two large breeding populations of bearded dragons were examined for the presence of I. amphiboluri oocysts using sucrose flotation and fecal direct saline smears. Parasites were more likely to be identified on fecal flotation (p= 0.03). A previously unidentified Eimeria species was examined and described as a new species for which we suggest the name Eimeria pogonae. To develop an understanding of the epidemiology of I. amphiboluri, characterization of the life cycle is needed. Fifteen I. amphiboluri negative dragons were used to determine the prepatent period, which ranged from 15-22 days. An additional 25 animals were infected with 20,000 sporulated oocysts to examine distribution in the gut. Animals were separated into five groups and euthanized at 4, 8, 12, 16 and 20 days post-infection and necropsy was performed. Infection began in the duodenum and move throughout the intestine. No extra-intestinal stages were found. Success with managing this parasite in captivity requires effective treatment. The first treatment study used three treatments, sulfadimethoxine, Pediococcus acidilactici, and oregano oil. Each group was treated for 21 days with 50mg/kg sulfadimethoxine, 10,000 CFU of P. acidilactici or 0.1 ml of Origanum vulgare essential oil. No significant difference was found in P. acidilactici, or oregano groups over time. There was a significant difference in sulfadimethoxine group over time (p=0.0001); however, the 21 day treatment was not sufficient in eliminating all of the parasites. The second treatment study evaluated ponazuril at three different doses. Each group was treated daily for 21 days with one of 4 treatments: isotonic saline, 15mg/kg ponazuril, 30mg/kg ponazuril and 40mg/kg ponazuril. All treatment groups showed a significant difference in shedding (p = 0.0001 each), suggesting that this drug could control the parasite in captive populations. I. amphiboluri is an important parasite of bearded dragons that has a moderate to high prevalence depending on husbandry methods. The combination of quarantine based on a 15-22 day prepatent period and treatment with ponazuril should be sufficient to control this parasite in captive populations.

Brucellosis, a zoonotic disease caused by Brucella spp., presents both health and economic difficulties for livestock, wildlife, and humans. While brucellosis is nearly eradicated in the United States, the disease remains detrimental in many countries worldwide. Attempts to produce a safe and effective small ruminant vaccine have been met with limited success. The current vaccine for bovine brucellosis in the United States is B. abortus RB51. This strain transiently colonizes the host and induces a cell-mediated immune response. Levels of protection have not been demonstrated in goats and thus it is considered a relatively poor caprine vaccine that probably does not survive long enough in the tissues to produce a sufficient protective immune response. This study analyzes the possibility of using RB51 containing plasmid QAE for vaccination in goats. The plasmid QAE contains a gene region of DNA from B. melitensis 16M hypothesized to encode for a putative hemagglutinin that is believed to be a host specificity protein. The region E gene sequence is not present in any strain of B. abortus, including RB51. It is proposed that the addition of region E to RB51 will enhance survivability in the caprine host to the extent where an adequate protective immune response is elicited. The QAE plasmid was electroporated into B. abortus RB51 and screened using ampicillin resistance as a selective measure. Ten goats were conjunctivally inoculated with RB51-QAE, the modified strain, and ten goats received strain RB51 in the same manner as controls. Two goats from each group were euthanized and necropsied at weekly intervals for four weeks and again at 42 days. Tissues from these animals were taken at necropsy and used to collect data for colonization and histopathology. Serum samples were also analyzed for Brucella-specific antibodies. Both strains transiently colonized the hosts without producing any detrimental pathology. However, the RB51-QAE goats demonstrated higher levels of colonization and greater humoral immune responses for longer periods of time. These are very promising findings as the levels of colonization and humoral responses may correspond with better protection. These results warrant further testing of RB51-QAE as a potential vaccine for caprine brucellosis.The putative hemagglutinin was characterized using hemagglutination assays, absorption assays, and mass spectrometry analysis following 2D gel electrophoresis. The region E protein was found to provide RB51-QAE with increased hemagglutinating and immunogenic capabilities. Furthermore, there is evidence to suggest that the protein is a cell surface protein, not excreted from the cell. The accepted gold standard for the detection of brucellae is bacterial culture. PCR is being evaluated as a possible alternative detection method for Brucella spp. in bodily fluids such as blood and urine. Currently, there is no accepted standard for detection of brucellae using PCR. The effects of template preparation, primer selection, and PCR optimization on the limit of detection for B. abortus 2308 and B. melitensis 16M in association with whole blood, plasma, or urine were examined. Ten-fold dilutions were made from a known number of bacterial cells in each of the fluids tested. The practices of using whole killed cells as a direct template for PCR as well as two differing DNA isolation techniques were evaluated for each fluid dilution. Our findings suggest that a more extensive template preparation technique and PCR protocol elongation can greatly improve the limit of detection capabilities. Biological fluids provided dissimilar results based on the PCR inhibition properties of the fluid and DNA isolation techniques. The results of this experiment encourage further investigation into the optimization of conventional PCR techniques as a faster and more efficient diagnostic tool for Brucella spp. in humans and animals.

Lyme disease is a multisystem disorder caused by the tick vector Borrelia burgdorferi. During its life cycle between the tick vector and the mammalian host, Borrelia up- and down-regulates the expression of its surface lipoproteins. Of its many surface lipoproteins, the Outer surface protein C (OspC) is crucial for initial mammalian infection. OspC has a common role shared with other lipoproteins of protection against host innate defences and a unique function in facilitating the dissemination of B. burgdorferi in the murine host. The structure of OspC was solved in 2001 and the lipoprotein was found to be predominantly alpha-helical with 5 alpha helices and 2 beta sheets interconnected by 6 loops, and an N- and C-terminus. Not much is known as to how the OspC structure relates to the function of the lipoprotein and/or possible regulation. The up-and down-regulation of OspC is achieved by the rpoS alternative sigma factor and by an unidentified repressor, respectively. Several truncated versions of OspC were tested in an OspC-deficient background to better understand the role of the amino acid sequences deleted. Structural deletions of 5 to 8-AA made within the OspC core, resulted in loss of infectivity. Longer deletions of 10- and 13 AA towards the N- and C-terminus of OspC, respectively, also resulted in loss of infectivity. Interestingly, a deletion of only 5-AA of the N-terminus of OspC did not affect the role of OspC in evasion but caused inefficient dissemination of B. burgdorferi in SCID mice. Interestingly, whereas the deletion of N-terminus 5-AA of OspC resulted in upregulation, a deletion of 6- and 9-AA of the C-terminus of OspC resulted in downregulation of ospC mRNA in joints, indicating that OspC may be involved in self-regulation. This hypothesis was confirmed by the use of monoclonal antibody treatment. Deletion of 6-AA of the C-terminus of OspC resulted in lower spirochete burdens in heart and joint, whereas deletion of 9-AA resulted in higher spirochete burden in skin, indicating the tissue-dependent effects resulting from the deletion of the C-terminal sequence. Taken together, these results indicate that OspC is involved in the pathogenesis of B. burgdorferi.

CD4+ memory T cells are depleted in mucosal tissues post human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) infection without restoration to pre-infection levels during progressive course of disease. Bone marrow (BM) as a hematopoietic organ has been investigated for hematologic and morphologic changes during HIV infection. However, BM as a primary lymphoid tissue during HIV infection has been poorly characterized. We proposed BM was also a site of CD4+ T cell depletion driven by increased apoptosis during progressive HIV disease. We chose to investigate bone marrow changes using the premier non-human primate Rhesus macaque SIV model for the study HIV infection. We observed hematologic abnormalities of anemia, thrombocytopenia, neutropenia and eosinophilia during SIV infection mimicked HIV infection as did morphologic increased BM cellularity, loss of iron marrow storage, and increased marrow fibrosis as infection advanced to AIDS. The increased BM cellularity was characterized by increased erythroid, myeloid including dendritic cells, and lymphoid lineages in the later stages of infection. In fact, numbers of BM CD3+ T lymphocytes increased in absolute numbers and proliferation percentage during progressive SIV infection mainly composed of CD8+ T cells and fewer CD4+ T cells. Naïve and memory CD8+ T cells and CD4+ T cells were maintained in BM during SIV infection. Low numbers of SIV infected BM cells were observed including CD3+ T cells, macrophages, and other hematopoietic cells with detection of both viral RNA and DNA by polymerase chain reaction. However, less than 0.2% of CD3+ BM T cells were apoptotic determined by activated caspase 3 though overall BM cells tended to have increased rates of apoptosis in later stages of SIV. Our data revealed BM T cells were not depleted but maintained to increased during SIV disease without an increase in apoptosis. Future studies into mechanisms of bone marrow lymphocyte maintenance may reveal homeostatic mechanisms potentially disrupted in mucosal tissues during HIV and SIV infection.

Francisella asiatica is a Gram negative, facultative intracellular pathogen that causes fish francisellosis. In this project, homologues to the F. tularensis iglABCD genes were found present in F. asiatica. As few as 23 F. asiatica bacteria injected in the peritoneum were found capable of causing mortalities in tilapia nilotica (Oreochromis niloticus), and even fewer were enough to cause pathological changes. We examined the in vivo and in vitro interaction of F. asiatica wild type (WT) and a ÄiglC strain with tilapia and tilapia head kidney derived macrophages (HKDM). The ÄiglC was found to be attenuated following intraperitoneal and immersion challenges in tilapia. The WT was found to be able to invade HKDM and replicate vigorously within them, causing apoptosis and cytotoxicity in the macrophages. The ÄiglC, however is defective for survival, replication and the ability to cause cytotoxicity in HKDM. We further characterize the efficacy of the ÄiglC as a live attenuated vaccine against subsequent immersion challenge with the WT. Tilapia vaccinated by immersion with a suspension of the ÄiglC and subsequently challenged with WT were protected (90% survival) from the lethal challenges. F. asiatica-specific antibodies produced in response to immunization with the ÄiglC were subsequently found to protect tilapia against WT challenge in passive immunization experiments. The lack of effective treatments led us to investigate the efficacy of florfenicol for treatment of F. asiatica in vitro and in vivo. Addition of florfenicol to the medium at 10 µg/ml resulted in uptake of the drug by HKDM and significantly reduced bacterial loads in vitro. Fish fed medicated feed 1 and 3 days post infection showed significantly higher survival rates, and significantly lower numbers of bacteria than controls. Finally, a real time polymerase chain reaction assay was developed to rapidly and accurately detect and quantify F. asiatica from fish tissue. Probe specificity was confirmed by the lack of signal and cross-reactivity with twelve common fish pathogens, two subspecies of F. tularensis, F. noatunensis, and tilapia tissue. The limit of detection was 50 fg of DNA (~25 genome equivalents).

Ticks are the only disease vectors for spotted fever group (SFG) Rickettsia which are obligate intracellular bacteria belonging to the genus Rickettsia. In nature, ticks maintain the infection of SFG Rickettsia via vertical and horizontal transmission. However, the prevalence of rickettsial transmission is limited to certain species of ticks, and this limitation is known as a specific tick/Rickettsia relationship. Due to the continuous increase of tick-borne rickettsial disease cases in the United States, which contrasts with very low prevalence of Rickettsia in tick vectors, the study of vector competence of tick to Rickettsia is needed in order to understand the ecology and epidemiology of tick-borne rickettsioses. Here we characterized the role of Dermacentor variabilis á-catenin during rickettsial infection in tick ovaries suggesting a role in rickettsial infection in tick ovaries. We demonstrated that the typical nonpathogenic (R. montanensis) and typical pathogenic (R. rickettsii) Rickettsia persistently infect Dermacentor variabilis compared to atypical Rickettsia (R. amblyommii), and only R. montanensis is able to disseminate to tick ovaries. D. variabilis glutathione S-transferase1 (DvGST1) has been identified as a tick immune-like molecule that specifically responds to atypical rickettsial challenge in tick midguts suggesting a role in controlling atypical rickettsial infection in tick midguts. DvGST1 is highly upregulated in tick midguts during bloodmeal acquisition. The function of GST is known to be involved with detoxification and oxidative stress reduction, and acaricide resistance in ticks. Silencing of DvGST1 gene demonstrates significant reduction of mRNA and enzyme activity of DvGST1 in tick midguts; however, further characterization of DvGST1 is needed due to the off-target effect of negative control dsRNA. Continued study on the tick/Rickettsia interaction influencing tick vector competence for Rickettsia will lead to a better understanding of ecology and epidemiology of tick-borne rickettsioses.